Article Figures & Data
Figures
- Figure 1.
The EMT gene expression signature separates NSCLC cell lines into distinct epithelial-like and mesenchymal-like groups independent of microarray platform. A, Affymetrix probes corresponding to the EMT signature genes were clustered by 2-way hierarchical clustering using Pearson correlation distance between genes (rows), Euclidean distance between cell lines (columns), and the Ward's linkage rule. NSCLC cell lines separated into distinct epithelial (green bar) and mesenchymal (red bar) groups at the first major branching of the dendrogram. Mutation status for EGFR and KRAS are indicated by the color bars above the heatmap (dark blue, mutated; light blue, wild-type; and white, unknown). EGFR mutations were seen only in the epithelial group. KRAS mutations were more common in the mesenchymal group and expressed higher levels of FN1 and FN1-associated genes. B, cell line classifications were concordant across platforms, with the exception of H1395, which switched from epithelial to mesenchymal group when arrayed on the Illumina WG v2 platform. First principal component analysis shows good separation of the epithelial and mesenchymal groups on both Affymetrix and Illumina platforms. C, characteristic differences in morphology are seen between lines characterized as epithelial or mesenchymal by the EMT signature. D, in an independent set of 39 NSCLC cell lines profiled on a third platform (Illumina WGv3), the EMT signature separated cell lines into distinct epithelial (green) and mesenchymal (red) groups by hierarchical clustering and principal component analysis.
- Figure 2.
Integrated analysis of protein expression and the EMT signature. A, RPPA analysis reveals that cell lines dichotomize into epithelial and mesenchymal groups based on their overall proteomic signature. B, E-cadherin protein levels quantified by RPPA were strongly correlated with the EMT signature first principal component in the training and testing cell line sets. C, hierarchical clustering of proteins strongly associated with an epithelial or mesenchymal signature showed higher expression of EGFR pathway proteins and Rab25 in epithelial lines. D, Axl expression was significantly higher in a subset of mesenchymal cell lines at the mRNA (using 2 different probes) and protein levels.
- Figure 3.
Mesenchymal lines are significantly more resistant to EGFR inhibition and PI3K pathway inhibition but sensitive to Axl inhibition by SGI-7079. A, relative IC50 levels of targeted agents are shown with P values corresponding to Wilcoxon rank sum test. B, fold difference between mean IC50 in epithelial (E) versus mesenchymal (M) cell lines. C, SGI-7079 inhibits Gas6-induced Axl phosphorylation as shown by Western blot analysis. Densitometry histogram of the Western blot analysis is graphed as the percentage of no drug treatment control of tyrosine phosphorylated Axl (p-Axl) relative to total Axl. The EC50 for SGI-7079 is less than 100 nmol/L. D and E, mesenchymal cell lines (red bars) are relatively more sensitive to SGI7079, whereas epithelial cell lines (black bars) are more sensitive to erlotinib. Gray bar (C) denotes 1 μmol/L concentration.
- Figure 4.
Axl blockade by SGI-7079 inhibits the growth of mesenchymal NSCLC xenograft tumors. A, mean tumor volume for A549 xenografts implanted in mice treated with vehicle or SGI-7079. B, mean tumor volume for A549 xenografts in mice treated with vehicle and the combination of SGI-7079 plus erlotinib.
- Figure 5.
Improved 8-week disease control in BATTLE patients with epithelial signatures treated with erlotinib. A, BATTLE patients (all treatment arms) were classified as mesenchymal or epithelial-like based on the EMT signature. B, among patients with wild-type EGFR and KRAS treated with erlotinib, 8-week disease control seemed superior in patients with more epithelial tumors (P = 0.052). C, there was no significant difference in 8-week disease control between epithelial and mesenchymal tumors in other treatment arms.
Tables
- Table 1.
Axl inhibition reverses EGFR resistance in mesenchymal cell lines
A549 Calu-1 H157 H1299 H460 H2882 Erlotinib IC50, μmol/L 13.54 >100 48.50 >100 >100 >100 SGI-7079 IC50, μmol/L 0.92 2.44 0.74 1.74 2.01 4.29 CI @IC50 0.46 >1.00a 0.67 0.72 0.57 >1.00a Combination: erlotinib + SGI-7079 IC50, μmol/L 1.07 + 0.35 13.86 + 4.47 1.46 + 0.47 3.76 + 1.21 3.53 + 1.14 16.44 + 5.30 -
↵aFor Calu-1 and H2882, the combination was antagonistic at IC50 but synergistic at higher concentration of SGI-7079.
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Supplementary Data
Files in this Data Supplement:
- Supplementary Methods - PDF file - 175K
- Supplementary Table 2 - PDF file - 51K, EMT status of commonly mutated genes in NSCLC
- Supplementary Table 3 - PDF file - 71K, Percentage tumor volume compared to control, survival and therapeutic efficacy at days 17 and 38 post-treatment
- Supplementary Figure 1 - PDF file - 167K, Different probes for the same gene vary within and across microarray platforms
- Supplementary Figure 2 - PDF file - 96K, Structure of pyrrolopyrimidine AXL inhibitor SGI-7079
- Supplementary Figure 3 - PDF file - 66K, CDH1 probes vary in their accuracy and dynamic range
- Supplementary Figure 4 - PDF file - 109K, EMT signature predicts resistance to EGFR and PI3K inhibitors and greater sensitivity to Axl inhibition
- Supplementary Figure 5 - PDF file - 50K, Mesenchymal lines are sensitive to Axl inhibition by SGI-7079
- Supplementary Figure 6 - PDF file - 116K, EMT signature predicts erlotinib sensitivity better than CDH1 or VIM probes. IC50 levels for erlotinib are shown
- Supplementary Figure 7 - PDF file - 27K, SGI-7079 and erlotinib do not adversely affect animal body weight
- Supplementary Table 1 - PDF file - 214K, The EMT signature genes
Volume 19, Issue 1
